反応 #586098

反応方程式

[3H]Dopamine

nicotine

→

Dopamine

反応条件

後処理

1. 1
   workup.WAITthe suspension was incubated at 37° C. for another 10 min
2. 2
   workup.WAITPerfusion buffer (room temperature) was pumped into the chambers at a rate of 3 mL/min for a wash period of 8 min
3. 3
   その他Fractions (12 sec each) were continuously collected from each chamber throughout the experiment
4. 4
   その他The perfusate was collected directly into scintillation vials, to which scintillation fluid
5. 5
   workup.ADDITIONwas added

実験手順

The synaptosomal suspension was incubated for 10 min at 37° C. to restore metabolic activity. [3H]Dopamine ([3H]DA, specific activity=28.0 Ci/mmol, NEN Research Products) was added at a final concentration of 0.1 μM and the suspension was incubated at 37° C. for another 10 min. Aliquots of tissue (50 μL) and perfusion buffer (100 μL) were loaded into the suprafusion chambers of a Brandel Suprafusion System (series 2500, Gaithersburg, Md.). Perfusion buffer (room temperature) was pumped into the chambers at a rate of 3 mL/min for a wash period of 8 min. Test compound (10 μM) or nicotine (10 μM) was then applied in the perfusion stream for 40 sec. Fractions (12 sec each) were continuously collected from each chamber throughout the experiment to capture basal release and agonist-induced peak release and to re-establish the baseline after the agonist application. The perfusate was collected directly into scintillation vials, to which scintillation fluid was added. [3H]DA released was quantified by scintillation counting. For each chamber, the integrated area of the peak was normalized to its baseline.

出典